1. Field of the Invention
This invention relates to an enzymatic analysis of endogenous glycerol and triglycerides in a sample to be assayed.
2. Description of the Prior Art
The measurement of serum triglycerides is important in the diagnosis of hyperlipoproteinemia and in the prediction, detection, and monitoring of atherosclerosis (1).
In the prior art, triglycerides are generally determined by a combination of hydrolysis to glycerol and free fatty acids and measurement of the amount of glycerol released. The most commonly used prior art methods involve alkaline hydrolysis and either chemical or enzymatic measurement of glycerol. Chemical means of analysis generally rely on measurement of the product of periodate oxidation of glycerol. Eggstein et al. (2) developed an enzymatic method for measuring glycerol released from triglycerides by alkaline hydrolysis. This method was based on the coupled reaction sequence catalyzed by glycerol kinase, pyruvate kinase, and lactate dehydrogenase. A method for complete enzymatic hydrolysis of triglycerides avoiding the need for serum pretreatment was described by Bucolo et al. (3) using a combination of lipase and at least one proteolytic enzyme. Wahlefeld (4) reported that certain esterases could be combined with a lipase to achieve complete hydrolysis of triglycerides. Both methods employed a coupled enzymatic reaction sequence (5) to measure glycerol.
Although there has been extensive research in the area of triglyceride analysis (6-16), none of the triglyceride analyses reported to date are capable of differentiating between the endogenous glycerol and endogenous triglyceride content of a sample without the necessity of having to perform a separate glycerol analysis on the sample being assayed. Instead, all of these prior art analyses include in their reported triglyceride content the amount of endogeneous triglyceride as well as the amount of endogeneous glycerol present in the sample being assayed. The reason for this can be seen from the schematic procedures set forth in FIG. 1. As shown in both of the schematic procedures of FIG. 1, the .DELTA. OD.sub.uv and .DELTA. OD.sub.c include a contribution from the assayed sample's endogenous glycerol content as well as the assayed sample's endogenous triglyceride content. In neither the ultraviolet nor colorimetric methods is it possible to add the lipase to the reaction mixture after obtaining a .DELTA. OD due solely to the sample's endogenous glycerol content. This is because lipase absorbs at both 340 nm and 520 nm and therefore would introduce an error into the resulting .DELTA. OD measurement. Therefore, in the prior art, if one desired to know the endogeneous triglyceride content of a sample, one would have to perform a separate glycerol analysis on the sample being assayed. The endogenous glycerol content found via this separate assay would then have to be subtracted from the data obtained using the prior art triglyceride analysis to yield the amount of endogenous triglyceride present in the sample.
It would be very desirable to have an assay which would be capable of determining the endogenous glycerol and endogenous triglycerides content of a sample without the necessity of performing a separate glycerol assay. Such a simplified procedure would entail the use of a single cuvette as opposed to the prior art necessity of employing at least 2 cuvettes. This simplified procedure would require less reagents as well as a smaller sample size than required to perform the two prior art assays. In addition, the simplified procedure could be readily automated, thereby effecting a major increase in productivity.